Monday, 10 March 2014

Rift Valley fever virus genome organisation: it's like it for a reason
Following recent Bluetongue virus and Schmallenberg virus (SBV) incursions into Central and Northern Europe, Rift Valley Fever virus (RVFV) is now perceived as one of the greatest threats to Europe, crossing over the Mediterranean from Africa where it remains endemic. With good reason. Both Culex  and Aedes species of mosquito are capable of transmitting the virus - West Nile virus infections in Italy have previously shown that there are conditions in Europe suitable for arbovirus transmission by such species of mosquito.
Like SBV, which has spread across Europe in the last 2-3 years, RVFV is a member of the Bunyaviridae family, (albeit in the genus Phlebovirus as opposed to Orthobunyavirus). If it were to encroach into Europe, the headline fact is that it's a zoonosis, with the potential to cause fatal disease in humans. Of equal, if not more, importance is the that fact that it can cause deaths and abortions in ruminants.

Dead cattle as a result of RVFV infection (

The RVFV genome comprises three segments of (-)ssRNA. Although the small (S) segment encodes both the nucleocapsid (N) protein and the non-structural protein NSs, a peculiarity is that, whilst N is produced from RNA transcribed from the genome, the message for NSs is transcribed from the antigenome. Superficially this is a little counter-intuitive; NSs is responsible for the rapid abrogation of the interferon system and thus would, presumably, be required as early as possible upon infection. Why force the virus to produce the antigenome before NSs can be produced?
A recent paper by Brennan, Welch and Elliott has addressed this by swapping around N and NSs, such that NSs is translated from the antigenome, and N from the RNA transcribed from the antigenome.

Generation of the swap virus: the (anti)genomic strand from which N and NSs are transcribed is 'swapped'.

In addition to the virus with wild-type (vaccine strain, MP12) and 'swap' virus, they also made viruses where NSs was substituted with EGFP. Now they had a panel of viruses including ones where NSs (or EGFP) were expressed first, before N. When they tested the growth of each virus, the swap viruses all grew poorly compared to the wild-type. This was regardless of whether the cells were either interferon competent (A549), incompetent (BHK21) mammalian cells, or indeed various insect cell lines.
Growth of 'swap' vs wt MP12 RVFV: In all cases, all swap viruses (filled circles) grow with lower efficiency compared to MP12 (A:mammalian cells; C. insect cells).

The lower apparent rate of replication was reflected in the amount of protein. As time progressed, the amount of protein accumulated by the swap virus was lower, although as expected the NSs protein was on this occasion produced before the N protein; opposite to what occurs with the wt virus. The accumulations of NSs were also much more substantial than with wt. However, although there was much more NSs (and less N) than wt, significant amounts of the glycoprotein (Gn) were not detected until 48 hours after infection, compared to 18-24 in the case of wt. In the case of the swap virus with EGFP in place of NSs, Gc was virtually undetectable even at 48h.
In wt RVFV, NSs assembles into filaments that are localised to the nucleus. Rather oddly, in the case of the swap virus, these were much thicker than the wt, with additional evidence of some NSs in the cytoplasm. These alterations in NSs behaviour appeared not to affect the functions of NSs in either inhibiting both host protein synthesis and host RNA synthesis. Given its 'shut-off' function, it is intriguing that increasing the abundance of NSs had no additional effect upon the intensity or rate at which protein shut-off occurs.  
Shut-off of host cell macromolecular synthesis: A: radioactive methionine/cysteine incorporation at different times post-infection; less label =  less protein = shut-off. B: shut-off of RNA synthesis (newly synthesised RNA is green, red is virus).

When they looked at the targets of NSs that result in shut-off, p62 was inhibitted by the swap virus (although more slowly), just as the wt but, bizarrely, PKR appeared to be largely unaffected by the swap virus, in contrast to the wt virus where PKR levels dropped from around 5 hours onwards. Overall, it would seem that, whilst it still does, the swap virus is a bit less efficient at the shut-off. As a result, it is a little surprising that the swap virus results in the induction of less IFN than the wt virus in A549 cells.

One thing the authors did tease apart is the relative number of genome:antigenome copies in both the cell and virion fractions. The swap virus was found to transcribe much more NSs RNA compared to the wt equivalent, suggesting that increased activity of the relative promoters is responsible for the dramatic amount of NSs observed with the swap virus. Such an excess of RNA may have overwhelmed the control by RNAi in insect cells, resulting in cytopathogenic effect in infected insect cells (in contrast to wt virus, which establishes a persistent infection). One interesting finding is that more antigenome than genome copies are packaged in swap virus virions. Does this reflect the abrogation a specific packaging process, or simply the abundance of genome:antigenome copies in the cytoplasm when packaging occurs? Considering the swap virus has N transcribed from the antigenome, the increase in antigenome packaging may actually overcome some of the temporal regulation achieved by swapping the N from the genomic to the antigenomic transcipt. It seems a bit more work  is required here. Virions have been shown to contain just 3 segments of RNA. If more of the virions have an antigenomic S segment, then the number of particles per infectious unit will necessarily be increased. It may be that such differences in the make-up of the viral population can explain some of the characteristics of the swap virus.

Of course such attenuating features - at least in vitro - may contribute towards the rational development of a vaccine. The most tempting feature of the swap virus described here is the fact that the virus cannot persist in insect cells and would thus be somewhat resistance to transmission; a key consideration in live arbovirus vaccines.

So, genome organisation is important. Ultimately this is not surprising: there's a reason it's like it is.

Brennan, B., Welch, S., & Elliott, R. (2014). The Consequences of Reconfiguring the Ambisense S Genome Segment of Rift Valley Fever Virus on Viral Replication in Mammalian and Mosquito Cells and for Genome Packaging PLoS Pathogens, 10 (2) DOI: 10.1371/journal.ppat.1003922

Sunday, 16 February 2014

Viral polymerases and pathogenesis
Read an introduction to viral evolution and you'll pretty quickly come across a sentence equating to 'viruses mutate and evolve fast and this is because their polymerases lack proofreading ability'. Sure, but that's a big generalisation. Large DNA viruses, for example, tend to have polymerases that are much less error-prone (again a generalisation). Nevertheless, in the world of RNA viruses it is a good rule of thumb that the polymerases tend to be error-prone when copying their genetic code. Superficially, such apparent laxity would appear to be detrimental to virus survival. However, if faithful replication of a nucleic acid was important, then the replication would indeed be faithful, but virus evolution occurs at a population level as opposed to the individual.

The viral sequences in GenBank are consensus sequences, in essence a read-out which represents the most commonly occurring base at each particular position in the genome. In a sense the concept of the consensus can be somewhat misleading: if RNA viruses have a mutation rate that is sufficiently great that it cannot copy a genome without making an error, then it's conceivable that the consensus per se does not, in reality, actually exist. Instead, a virus is a population of sequences evolving together, exploring sequence space with a great level of diversity.
Virologists tend to roll out the term quasispecies to describe this, but there remains some debate as to the validity of this theory, certainly in relation to Eigen's initial proposal.

Survival of the flattest: when mutation rates are low, all viruses accumulate together; if the peak is steep and narrow, then a low mutation error results in all of the viruses being exceptionally fit, (A outcompetes B). If mutation rates are high, then fitness is spread, in which case a spread of highly fit viruses is preferable to an extremely fit virus surrounded by viruses of low fitness (B outcompetes A, from Wilke 2005). 
Having such diversity means that the virus can tackle things that get in the way of progress, most obviously an immune response; if the response impacts upon the fitness of one sequence, then there's another that is capable of preserving the virus' existence. In order to generate such diversity, a virus must necessarily make mistakes and thus viruses tend to live quite close to the maximum permitted error rate for a particular lifestyle. Too much error, and the virus suffers error catastrophe and becomes extinct, as there are insufficient fit sequences. Too little error though, and sufficient diversity becomes hard to generate.

A paper came out recently describing a variant of Chikungunya virus (CHIKV) with altered levels of fidelity. A CHIKV mutant had previously been isolated by growing the virus in the presence of the antiviral ribavirin, which resulted in a virus with increased fidelity. This virus had a single mutation in the viral polymerase. The authors systematically changed this amino acid and looked at the effect it had. Of the 19 options attempted, 12 viruses were viable.

Figure 1 Mutagenizing position 483 variants allows isolation of mutator variants.
Mutator variants: A. the impact of different amino acids on the susceptibility to ribavirin treatment. B. mutation frequencies of selected variants compared to wild-type virus; G and W result in increased rates of mutation. C. average diversity of confirmed fidelity variants at each point across the genome; anti-mutator variant with a Y results in lower diversity across the genome. D. due to the greater diversity, mutator strains are better able to escape virus neutralisation by antibody (CHK-102).  
If these viruses have altered fidelity, then they should have different sensitivities to ribavirin, and indeed some viruses were less sensitive to ribavirin (antimutator strains) whereas some were more sensitive to ribavirin (putative mutator strains). When they checked for sequence diversity, they found a matching trend that sensitivity to ribavirin correlated with diversity, i.e. mutator viruses that make mistakes are susceptible to the treatment. A more highly mutated genome would in theory result in more non-viable/unfit genomes, and indeed they found that the progeny virus population of mutator strains was less infectious, even though their replication in mammalian cells was largely unaffected. When they injected some of the viruses into mice, there was a correlation between the frequency of mutation and viral loads. In line with the in vitro data, the mouse model revealed a decrease in pathogenicity associated with the increased error rates of mutator strains.

Whilst mutator strains replicated fine in mammalian cells, they replicated poorly in mosquito cells. As might be expected under such pressure, in vivo infections of mosquitoes, as well as passage in insect cells, resulted in a reversion of the mutator strains to a more wild-type level of fidelity. It would be interesting to see whether these viruses could be transmitted to subsequent hosts via mosquito bites.

Mosquito infections: Aedes  albopictus (A-C) or Aedes aegypti (D-F) mosquitoes were fed blood containing wild-type (WT) or mutator versions of CHIKV. Virus levels of all viruses was equivalent in the 

Another way in which they confirmed the impact of the changes associated with the polymerase fidelity and its impact upon virus phenotype was to specifically alter the equivalent position in another alphavirus: Sindbis virus (SINV). When they tested the mutated version of SINV, they found the same outcome, notably relatively little impact upon replication levels in mammalian cells, attenuation of pathogenicity in mouse models, lowered titres in insect cell cultures, and reversion to 'wild-type' in mosquito infections, thus confirming the importance of the mutator phenotype.

Mutator (and antimutator) viruses such as these may offer ways in which we can study further how viruses live close to the error threshold - perhaps with the prospect of adding evidence for or against the 'quasispecies' concept.
But replication competent virus but with attenuated phenotype? This rings the vaccine bell. At least potentially. There's more to do, and the authors highlight this, in particular with regard to how the viruses under selective pressure in vivo will behave and evolve in different hosts. But they're certainly an intriguing prospect.

Rozen-Gagnon, K., Stapleford, K.A., Mongelli, V., Blanc, H., Failloux, A-B., Saleh, M-C., Vignuzzi, M. (2014). Alphavirus Mutator Variants Present Host-Specific Defects and Attenuation in Mammalian and Insect Models PLOS Pathogens DOI: 10.1371/journal.ppat.1003877

Wednesday, 22 January 2014

Dengue in Viet Nam
In some, maybe the majority of cases, economic development tends to improve a country's situation regarding infectious disease; a proper sewerage system, for example, may decrease the incidence of diseases associated with the contamination of water sources. Where dengue virus is concerned this is not the case. Dengue is a human virus spread by mosquitoes of the genus Aedes (in particular A. aegypti), which happily breed in dirty water. Economic development tends to result in increased urbanisation and, as a result, ideal breeding conditions are generated for the mosquitoes (tin cans, old tyres etc.). Together, the result is a dense population of humans in the same location as the mosquitoes: in the absence of a vaccine or antivirals dengue has thus thrived. Whilst the conditions are favourable for dengue in general, there are inevitably more specific drivers of transmission and outbreaks.

A recently published study in PLOS Neglected Tropical Diseaes by Rabaa et al looked into what the drivers are for dengue in Viet Nam. The situation in Viet Nam can broadly be regarded as the south (tropical) region being endemic, whilst the north (sub-tropical) is not endemic, but experiences frequent introductions. In central Viet Nam the virus can persist for more extended periods of time, perhaps due to more favourable conditions for transmission and, ultimately, a higher level of immunity. As an illustration as to the impact of urbanisation, Ho Chi Minh city in the south is highly endemic and represents a large source of viruses for the rest of Viet Nam.

The authors compared the dengue serotype 1 (DENV-1) sequences of the envelope (E) gene.
Using a maximum likelihood approach to get an additional grasp of geographical relationships, they found that, perhaps unsurprisingly, all of the sequences belonged to the Southeast Asia subtype of Genotype I.

Phylogeography of DENV-1 genotype 1 in Southeast Asia, 1998-2009. A) Map of north (red) central (yellow) and south (blue) Viet Nam. The colours match in the graphs of mean min/max temperature (B, top graph) and mean precipitation (B, bottom graph), and branches in the maximum clade credibility phylogenetic tree (C). Purple branches represent Singapore sequences.

On the whole, DENV-1 seems to invade subtropical northern Viet Nam regularly, but never seems to become endemic - most likely due to the cold winter temperatures resulting in conditions that are refractory to continued transmission. Such invasions also occur in the central regions, although these persist for longer.
Interestingly, although (as may be expected) within a particular region the diversity among viruses was limited,  on a broader scale Ho Chi Minh City in the south was found to act as a source of virus throughout Viet Nam.

On the other hand, despite local diversity being low, it's interesting that geographically long distance movements were observed in a time-scale that precludes the hypothesis that it's merely natural spread via vectors. Instead, it appears that the movement of infected humans is responsible for seeding at least some of the regions. This is one route by which the north can be seeded. However, because the north is sub-tropical, there comes a time in the year when the vectors die off and transmission is reduced; a familiar scenario with non-tropical arboviruses. 

As interesting a piece of work as this is in itself, it arguably demonstrates something important at a more global level. Clearly DENV can be seeded in different regions by people moving around Viet Nam; if this can happen within Viet Nam, then it's not a massive step to extend Viet Nam to the world.

Maia A. Rabaa, Cameron P. Simmons, Annette Fox, Mai Quynh Le, Thuy Thi Thu Nguyen, Hai Yen Le, Robert V. Gibbons, Xuyen Thanh Nguyen, Edward C. Holmes, John G. Aaskov (2013). Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance PLOS Neglected Tropical Diseases DOI: 10.1371/journal.pntd.0002581

Friday, 3 January 2014

Bats vs. Rats
If I had to name one book that got me interested in viruses it would be 'Virus X: understanding the real threat of the new pandemic plagues', by Frank Ryan. The book largely concentrates upon virus emergence; why it happens, where the viruses come from, and what that might mean for the future. Whether or not I now agree fully with everything that's hypothesised is a different matter, although an honest evaluation is difficult considering the advances in science since its publication (1997). Nevertheless, it got me interested at the time.

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Flicking back through it last night I read a line regarding virus reservoirs that stood out. "The threat to humanity derives in particular from rodents". This was the logical conclusion derived from the fact that rodents are the most numerous mammal, which is fair enough. Nowadays it's almost all about bats. In the book Ryan does point out the suspicions of bats as reservoirs, but overall in this book the potential significance of bats is over shadowed by a focus on rodents.

With our current knowledge of virus natural reservoirs (a term itself worthy of debate), a suggestion that anything other than bats are the most important source of viruses as far as public health is concerned, is likely to be met with an element of scorn. Bats do indeed harbor a lot of viruses, as was published earlier this year. In this particular paper the authors also estimated the numbers of viruses still to be discovered (320,000), although wisely they also stated that such a calculation was based upon some rather large assumptions:

"Several important limitations must be considered in our extrapolations, including (i) the assumption that a mean of 58 viruses per species is a reasonable estimate and that host populations are panmictic with respect to viral transmission (such that expanded geographic sampling would not influence viral detections), (ii) the assumption that viruses are not shared by more than one host species, (iii) that only those viruses within the nine families are considered in this estimation, (iv) that the results are limited by the sensitivity and specificity of our tests, and (v) that a similar mean cost of sample collection is incurred across all species."  (Anthony et al. 2013, mBio) 

Nevertheless, it's a useful number to have.
Bats have long been suspected as reservoirs, and in the case of rabies it had been firmly established, but I'm not sure when exactly they became so popular for virus hunters. Perhaps around the time of Nipah and Hendra emergence. Nowadays everybody seems to be hunting for viruses in bats specifically.

Bats, it can safely be said, represent an important source of novel as well as known viruses. In terms of virus emergence and spread however, there is more to it. Yes, bats may harbor a lot of viruses. And perhaps yes for one reason or another those viruses may have a higher chance of being unpleasant. But there is more to epidemiology than simply the source. Spillovers in a forest/rural setting are inevitable, and in this case bats pose as much of a risk as rodents. However, the majority of people live in urban areas. And from this perspective, rodents are surely of greater importance for transmission as their populations are so intimately linked with humans. More contact means a greater likelihood of transmission. One of the worst epidemics in history, the Black Death (admittedly caused by a bacterium), was closely linked with rats. More recent, viral, examples include the Sin Nombre hantavirus in New Mexico, and the Arenaviruses (e.g. Lassa fever virus).

It could be argued that, because we've had so much interaction with rats over the years we're unlikely to find anything new. That doesn't mean they're of lesser importance; Lassa fever and Sin Nombre are responsible for the death of more people than those caused by more exotic viruses such as Ebola virus.

Global air travel: 'emergence hotspots' such as South East Asia experience more international travel than central Africa. Image: Max Planck Institute for Dynamics and Self-Organisation/ Dirk Brockmann
It is clear that the jungles and savannas of Central Africa harbor bats with viruses of great danger to humans. But is this more important than, say, the populations of rats in densely populated urban centers in South East Asia? Human traffic to and from such urban areas is higher, enhancing the probability of an infection spreading to other parts of the world; would SARS, for example, have spread so far if it had emerged in Uganda? In the future it may be that the world is equally connected. For now though, some places remain more connected than others, and this should be remembered when people decide what's more important: bats or rats.

Anthony SJ, Epstein JH, Murray KA, Navarrete-Macias I, Zambrana-Torrelio CM, Solovyov A, Ojeda-Flores R, Arrigo NC, Islam A, Ali Khan S, Hosseini P, Bogich TL, Olival KJ, Sanchez-Leon MD, Karesh WB, Goldstein T, Luby SP, Morse SS, Mazet JA, Daszak P, & Lipkin WI (2013). A strategy to estimate unknown viral diversity in mammals. mBio, 4 (5) PMID: 24003179

Monday, 18 November 2013

Should I have published?
Since finishing my PhD I've been faced with a dilemma. In a nutshell, having come across a (somewhat serendipitous) observation in my PhD studies, should I publish it? I decided to publish, and it was both an editor's pick and is now regarded by the journal as 'highly accessed' (the importance and possibly ephemeral nature of such labels is a completely different discussion). That implies it was worthwhile, but was it?

The study revolved around an observation in BHK (hamster) cells infected with Bluetongue virus (BTV). The cells looked very strange: rounded and with condensed DNA/chromosomes in a pattern suggestive of some stage in mitosis, albeit a rather odd looking mitosis. To try and see what's going on, we used confocal microscopy with a panel of antibodies to look at the status of various parts of the cell division machinery. In brief, we found that the centrosome, a major orchestrator of mitosis, was severely disrupted. Co-incidence or not, the BTV protein non-structural protein NS1 also located in the region.

A-D. Different BTV serotypes (16, 1 and 8) induce aberrent mitoses (although BTV-16v induces the most). Different cell types can also be affected, although BHK cells appeared to be the most susceptible.
Something that was conspicuous was the association of the viral NS2 protein with the condensed chromosomes. When we took a series of images in the z plane and analysed them it became clear that NS2 appeared to be associated with the kinetochore. Combined with the observation of its location on microtubules, it is conceivable that NS2 may be a microtubule cargo molecule (or interacting with one) that obscures the kinetochore during the initial stages of mitosis. As the microtubules polymerise though the cell, the tips don't find the kinetochore, resulting in faulty mitosis. Many viral proteins use microtubules to get around and, based on other viruses, the dynein/dynactin complex would be an interesting  place to start looking for a protein that interacts with NS2.

A. NS2 expressed from a plasmid locates to microtubules (red). B. Z stack images reveal NS2 located at positions suggestive of the chromosome centromeres. C and D. Expression of NS2 from a plasmid recreates the aberrent mitotic phenotype.

To look at whether NS2 alone is capable of inducing the aberrent mitosis, we transfected cells with plasmids encoding the protein. When looked at from a confocal perspective, the transfected cells appeared to reflect the phenotype seen with virus infection. When a GFP-tagged version of NS2 was used in live cell imaging, we found that the cells were less likely to complete mitosis correctly, spent longer in mitosis, and resulted in an increased level of binucleated cells.

Transfecting HeLA cells with a palsmid expressing a GFP-tagged version of BTV NS2 resulted in a longer time spent in mitosis, a reduced level of successful mitosis, and binucleation.
 So, to the options. 
1) don't publish. At the end of the day it's just an observation; I have not elucidated an exact mechanism and nailed down a precise protein, as would be expected for a publication in a journal of greater 'impact'. Not taking the story to an end, followed by publishing in a prestigious journal might be viewed as poor science by some. 
2) publish. Many would argue that publishing information, regardless of how seemingly insignificant, is important and, arguably, a necessity based on the fact that it is being funded by the public.

I published. Partly for the reasons outlined in scenario 2, but also because the study was at a point where other people had contributed work, in which case it would not be fair for them to have done this work only for me not to publish. Of course, continuing the project to the end would have been my (and my collaborators') preferred option, but time ran out. As it stands, this observation is in the public domain for all to see, with the option of progressing it further to try and unravel what's happening.

Should I have published? I'm satisfied that I did, but it once again highlights the question of how many other such observations are languishing in abandoned lab books around the world.

Andrew E Shaw, Anke BrĂ¼ning-Richardson, Ewan E Morrison, Jacquelyn Bond, Jennifer Simpson, Natalie Ross-Smith, Oya Alpar, Peter PC Mertens and Paul Monaghan (2013). Bluetongue virus infection induces aberrant mitosis in mammalian cells Virology Journal DOI: 10.1186/1743-422X-10-319

Sunday, 10 November 2013

Down on the farm with Schmallenberg virus: the full story
I've already written a couple of posts about Schmallenberg virus (SBV), a bunyavirus that emerged in Northern Europe in 2011. What I haven't discussed is the SBV experience on my family's diary farm. Clearly an opportunity not to be missed, this has just been published. The thing that scientific publications can't convey however is the meandering thoughts and subjective observations that have little or no scientific rigour. A scientific manuscript can only report concrete and measurable results. Hence this post.

SBV is difficult to spot as the most dramatic clinical signs tend to be malformed offspring, which is a somewhat rare occurrence, at least in cattle herds. As a result there is a period whereby the virus may have been around for several months before it is discovered: when we first found SBV on the farm, the UK was more or less at this stage. In February 2012 there were only a few reported cases of SBV, all around the SE fringes of England where SBV-laden Culicoides had presumably been blown across the channel.

No doubt at least partially as a result of my ongoing comments on the phone about the SBV situation, when a cow oddly aborted close to term, "could it be SBV?" was a question that immediately arose. The cow, number 157, along with some others, was bled and the samples sent to me in Glasgow, where I tested it for SBV antibodies. The result was a clear positive for SBV.

The first ELISA result of SBV on the Bishops Farm. C2 and D2 = cow 157. A4-D4 = positive control.
And was positive again when a second sample was taken.

Another way in which to determine whether #157 was positive for SBV antibodies was to immunolabel some cells infected (or mock infected) with SBV. Serum from #157 clearly detected SBV whereas serum from the other animals didn't react.

(a) the s/p output values from the antibody ELISA of the cows tested following #157's abortion. When serum from #157 was used to immunolabel cells, green signal was only seen in cells infected with SBV.

SBV was present on the farm. At the time, this was several degrees further north than the known distribution. We tested to see whether the antibodies that were recognising the virus were IgM isotype (in which case the infection was recent) or IgG, meaning that the infection was older. Everything was IgG, so the infection had been around for a bit. How long had it been in the area?

In essence this was a 'just in time' scenario; soon virtually all of the UK's farms would be positive for SBV. I found it hard to believe that vet schools weren't already screening their flocks and herds, but here was an opportunity to look at seroprevalence at the herd level in a 'typical' UK commercial dairy farm. So every animal in the herd was sampled and tested for SBV antibodies. An important aspect is that no animals were moved onto the farm during the previous months, therefore the SBV must have arrived by some other means - clearly the likely option being midges. Only a few of the herd were positive, but clearly SBV was present.

During the summer two deformed calves were born. In over 20 years previously, only a single deformity had occurred in this herd. What's more, the dead calves had issues with joints, something that would be consistent with the deformities observed with SBV. One in particular had features that looked extremely similar to those in the literature that had been confirmed as having SBV, including fused and stunted limbs.

A dead calf with clinical features sugestive of SBV, including stunted and fused joints, most obviously a suggestion of arthrogryposis in the hind legs.

The other calf seemed quite the opposite, as if there were no joints, resulting in a floppy carcass, even if the calf otherwise (outwardly) looked fine. 

A deformed calf born in the summer, with a 'bag of bones' type deformity.

Two deformed calves, knowing that SBV had been present at the crucial time, certainly seemed suggestive that these deformities were as a result of SBV. But this isn't in the paper as we can't state that they were the result of SBV without testing them for the presence of the virus. A post mortem would have been revealing.

Another thing that is rather superficial in the paper is another aspect often associated with SBV, changes in milk yields. Overall there was a depression in the milk yield, but there's no way of proving that this was not because of some other factor. What was more dramatic were sudden acute periods of no milk combined with what appeared to be severe depression, but again it's impossible to say that this was as a result of SBV. If we'd tested these animals and it had coincided with SBV viraemia, then perhaps we could say it was related. In reality, these acute episodes are the most commonly observed clinical feature of SBV, at least in cattle, with the deformed offspring representing the exception rather than the norm - there were many other calves born that were perfectly fine. Somewhat frustratingly it is the deformities aspect that people most want associated with SBV, thus that's what dominates in SBV papers and talks, including this paper. It's difficult to nail this kind of thing down though as everything is by association rather than causation, and in many cases is difficult to measure, e.g. how do you know a cow is feeling rough due to SBV? 
In the paper there's a mention of high levels of diahorrea. Again, this is difficult to a) quantify and b) inextricably link to SBV circulation in the herd.

When it got colder, and the midge season was theoretically over, we tested again. This time the majority of the animals were positive for SBV antibodies. Clearly SBV had spread throughout the herd over the summer period.

The proportions of animals in the milking herd that were positive for SBV either before or after the summer period.

This is not in the least surprising. A more dramatic result would have been if there had been any other outcome. The interesting fact though is that, during the summer, the milking cows were at pasture for only a few days. Dogma until a few years ago was that midges are generally reluctant to enter livestock housing. This was based primarily upon observations in the field of Bluetongue virus (BTV), where the Afro-Asiatic species C. imicola is the key vector. It is now established that European midges are perfectly happy to enter buildings. As well as exposure to vectors, another key driver of arbovirus transmission is temperature, which affects both the biting behavior of the vector, and also the kinetics of virus replication within the midges. When the first case in #157 was found, the temperature was only around 10 degrees. The obvious caveat here being that this temperature is the outside temperature; inside it's warmer - that temperature would be very interesting to know. This is relative though: it may be warmer inside but that's relative to 10 degrees; even if it was 5 degrees warmer that's still only 15 degrees. This is still quite cool.

Overall the message would seem to be that not letting the animals spend their days and nights roaming freely at pasture is no barrier to arbovirus transmission. This perhaps shouldn't be surprising. It's warmer, the breeding habitat is textbook for Culicoides, the animals are closer together, there's no wind etc. and there is, inevitably still exposure to the outside.
I'm clearly biased, but the beauty of this study remains that it reflects a real situation. This is not a controlled vet school farm. It is not a sentinel herd kept to check for the first incursion. It is a working dairy farm that more accurately reflects the average scenario for the UK.

And lastly, in case you wondered, #157 has a name: Blossom.

A. E. Shaw, D. J. Mellor, B. V. Purse, P. E. Shaw, B. F. McCorkell, M. Palmarini. (2013). Transmission of Schmallenberg virus in a housed dairy herd in the UK Veterinary Record DOI: 10.1136/vr.101983

Monday, 7 October 2013

Filming fluorescent Marburg virus
For some Marburg is a city in Germany. It's also the name of a virus closely related to the much more widely known Ebola virus (a name which people tend to associate with a virus as opposed to the small river it's named after). What they both have in common, beyond both being members of the Filoviridae family,  is a propensity to induce highly unpleasant, and often lethal, haemorrhgagic fevers. Marburg virus (MARV) first surfaced in 1967 in laboratory workers in Marburg and Yugoslavia and, just like Ebola, has caused sporadic cases and outbreaks since then, the most horrifying of which was in 2004-2005 in Angola, where 227 of 252 (90%) of those known to be infected died.

The worm-like form of Marburg virus particles. 
As per many viruses, much about the MARV lifecycle within the cell remains a mystery. A recent paper in PNAS used live cell imaging to dissect some of the events involved in making new viruses and how they shuttle to a point of release. 

Live cell imaging is often based upon fluorescence, so one of the first things was to make the tools. Essentially, they made versions of structural viral proteins, VP30 and VP40, that are tagged with a fluorescent molecule. To VP30 they added green fluorescent protein (GFP) and showed that when expressed from a plasmid it behaved like the untagged VP30. Similarly, they inserted a red (RFP) version of VP40 into the genome of the virus, such that wild type (wt, = unmodified) and tagged VP40 were produced. The new virus behaved similarly to the unmodified virus, at least early during infection. In infected cells, RFP-VP40 colocalised with wt VP40, implying that this modification didn't alter its localisation. Tools made.

The first step of virus production/release they looked at was the exit of nucleocapsids from the inclusions where nascent viruses are thought to assemble. When they filmed inclusions, VP30-GFP was seen to be leaving, confirming this is where nucleocapsids are assembled, but not with RFP-VP40 (despite VP40 being present at the inclusion body), leading to the conclusion that VP40 is added elsewhere.

Nucelocapsids leaving the inclusion. Individual nucleocapsids could be seen leaving (top) and of those leaving, VP30 but not VP40 was present (bottom) 

If the VP40 component of particles is being added somewhere other than the inclusion, then the obvious question to ask is 'where does VP40 get added?'. Again, they turned to microscopy. When they counted the number of nucleocapsids containing both VP40 and VP30, the number increased towards the plasma membrane, implying that it is here that VP40 associates with the nucleocapsids.

VP40 gets added near the plasma membrane: the closer a nucleocapsid is to the plasma membrane, the greater the likelihood that it also contains VP40, suggesting that it is at the cell periphery that VP40 becomes associated with nucleocapsids.

A  bonus of filming an infection is that there is an additional parameter, i.e. time. This means that the speed at which things happen can be worked out. In this case the authors were able to work out that the nucleocapsids moved at up to 500 nm/sec. On top of that, they were able to figure out that the movement was quicker towards the centre of the cell, as opposed to more remote regions, possibly because nucelocapsids are using different motor proteins in different regions to surf the cytoskeleton. But which component of the cytoskeleton? Two approaches were used. First, they filmed VP30-GFP labelled nucleocapsids in cells with either red tubulin or red actin: only in the case of actin was the movement consistent with riding a particular filament .

In the second approach, treating cells with nocodazole, which disrupts microtubules, had no effect whereas cytochalasin D disruption of the actin filaments brought MARV movement to a halt. In both cases actin appears to be the answer.

Lastly, they looked at the presence of the nucleocapsids in the filopodia extruded from the infected cells. From their observations, they concluded both that VP40 must be associated with the nucleocapsids, and that the motor protein Myosin 10 (Myo10) is involved in the transport of nucleocapsids in the filopodia.

This work is impressive for many reasons. most immediately obvious is the reliance upon live cell imaging. Very often there are the (reasonable) requirements for observations in the microscope to be backed up by biochemical data. It's a great example of what can be achieved via deductions made from observations in careful experiments. The difficulty in doing this work is also easy to overlook. I get the impression that doing this project in BSL-4 would be tricky. Conveniently, they had a remote controlled microscope that they could operate from a more comfortable location, something that's rather handy if you're going to film fluorescent Marburg.

Gordian Schudt, Larissa Kolesnikova, Olga Dolnik, Beate Sodeik, and Stephan Becker (2013). Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances Proceedings of the National Academy of Science DOI: 10.1073/pnas.1307681110